DESCRIPTION: Injectable biopharmaceuticals produced in current systems are prohibitively expensive and are not affordable for a large majority of the global population. Recent studies have shown that bioencapsulation of protein drugs in plant cells are protected in the stomach. Commensal microbes play a critical role in their release into the gut lumen. Rapid delivery of drugs bioencapsulated plant cells into circulation, histological evidence and studies with antibiotics show the critical role played by small intestinal microbes in this process. We have also identified unique tags that could deliver therapeutic proteins to the circulation, non-immune or immune modulatory cells after crossing the gut epithelium. This proposal addresses some of the remaining challenges in oral delivery of human blood proteins so that this novel concept could be advanced to the clinic. Protein drugs should be expressed in edible plant cells instead of tobacco and their expression level should be enhanced (especially for large proteins) to facilitate dose escalation studies. In order to achieve this, cutting edge modern tools will be utilized. Ribosome profiling of native human genes expressed in chloroplasts will identify ribosome pause sites. Codon optimization will substitute rare codons with optimal codons. Level of expression of GLP-1 like short peptides (exendin) or angiotensin 1-7 will be enhanced with tandem repeats and cleavage sites with ubiquitous proteases like furin. Different delivery tags will be tested to target proteins to non-immune or immune modulatory cells and their functionality will be evaluated for treatment of hypertension/diabetes or induction of oral tolerance in hemophilia using suitable animal models. The role of gut microbes will be studied by functional characterization and identification of human small intestinal bacterial species capable of releasing protein drugs bio encapsulated in plant cells and host absorption. Production successful candidates in cGMP facility (Fraunhofer USA, Delaware), evaluation of stability upon prolonged storage at ambient temperature, efficacy and dosage are proposed. These novel approaches should improve patient compliance in addition to significantly lowering the cost of healthcare by elimination of prohibitively expensive fermenters, purification, cold storage/ transportation, sterile delivery of currently used methods and extending shelf life of protein drugs. Clinical advancement of this concept would revolutionize protein drug production and delivery for most metabolic and genetic disorders. In contrast to the well-studied fecal/colonic system, functional characterization of the human small intestinal microbiota has not been previously explored and this study would facilitate further mechanistic understanding of the human gut and the role of intestinal microbes in processing of plant cells and delivery of protein drugs utilizing the largest absorption surface in the human body.